Cloned CD4-CD8-alphabeta murine T cells can be induced in vitro to secrete a novel 20 Kd cytokine Natural Suppressor Factor (NSF) which inhibits IL-2 secretion of T cell hybridomas or fresh murine T cells stimulated with antigen but not mitogens. The cytokine has unique N- terminal as well as internal amino acid sequences, and inhibits IL-2 secretion by interfering with the function of antigen presenting cells (APC's). The main goal of this proposal is to clone and express the gene which encodes the cytokine, such that a recombinant cytokine can be produced which can be used for further in vitro and in vivo analysis of its biological functions. Oligonucleotide probes based on the unique N- terminal and internal amino acid sequences will be synthesized and used to screen a cDNA library from a cloned CD4-CD8-alphabeta T cell line. Candidate cDNA clones will be transfected into COS-7 cells, and supernatants will be tested for the presence of appropriate biological activity, as well as binding to specific monoclonal and polyclonal antibodies using ELISA and Western blot analysis. The biochemically purified and/or recombinant cytokine will be studied for the capacity to reduce APC secretion of IL-1, IL-6, and TNFalpha, and APC expression of surface molecules (MHC Class II, ICAM-1, LFA-3, B7) which interact with T cells. The cytokine will be studied as well for the capacity to reduce IL-2 secretion and to induce anergy in T cell clones in vitro including a myelin basic protein (MBP) reactive clone which induces experimental allergic encephalomyelitis (EAE). The function of the latter clone will be evaluated both in vitro and in vivo. Monoclonal antibodies to the cytokine will be produced also so that a sensitive ELISA can be developed.